Baker’s yeast Saccharomyces cerevisiae has been modified by metabolic engineering to ferment the pentose sugar xylose. Xylose is commonly found in forest and agricultural residues and is important for process economics of ethanol production from lignocellulose biomass. Recombinant xylose-utilizing Saccharomyces cerevisiae however lack specific transporters for xylose. Consequently this substrate enters the cell through non-specific hexose transporters. Compared to glucose, these transporters have poor affinity for xylose, and it has been hypothesized that this step may control the rate of xylose utilization. Furthermore, the high affinity hexose transporters responsible for xylose uptake, show significant repression by glucose during co-fermentation of glucose and xylose. To improve xylose uptake and reduce repression by glucose, we have expressed the previously identified xylose transporters Gxf1 (Candida intermedia), Sut1 (Piccia stipitis) and At5g59250 (Arabidopsis thaliana) in recombinant Saccharomyces cerevisiae. Resulting strains were characterized in terms of xylose transport kinetics following cultivation in different media. The strains were also compared in aerobic and anaerobic batch cultivation at different substrate concentrations. Results are discussed in relation to different industrial fermentation techniques and the composition of substrate feeds.